Salt-stable complexes of the Escherichia coli RecBCD enzyme bound to double-stranded DNA.
We have examined binding of the RecBCD enzyme to linear double-stranded DNA under two types of conditions. Binding in the absence of ATP can be measured by a nitrocellulose filter binding assay or by gel retardation on polyacrylamide gels. The binding is tightest to ends with four-nucleotide single-stranded 3'-termini (3'-overhang > 5'-overhang > blunt ends). The Kd for blunt ends is 3. 0 (+/-0.5) nM, in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2. The binding is weakened in the presence of NaCl, with none detected at 0.5 M NaCl. Binding in the presence of ATP and low MgCl2 concentrations ("unwinding conditions") can be measured by the filter assay and on agarose gels. Enzyme-DNA complexes allowed to form under unwinding conditions are not affected by 0.5 M NaCl. ATP hydrolysis continues and the complexes dissociate at a rate similar to those to which no salt is added. These enzyme-DNA complexes can be trapped with EDTA, and they are unaffected for at least 1 h by 0.5 M NaCl or heparin. The results show that the enzyme-DNA interactions are different when the enzyme is bound to partially unwound DNA compared to when it is bound to the ends of fully duplex DNA.
Gabbidon
M R
MR
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Rampersaud
V E
VE
Julin
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D004268:DNA-Binding Proteins; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D006493:Heparin; D004247:DNA; D004492:Edetic Acid; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004265:DNA Helicases